Il 4 pe cyanine7

Manufactured by Thermo Fisher Scientific

Sourced in United States

IL-4-PE-Cyanine7 is a fluorescent conjugate used for the detection and analysis of interleukin-4 (IL-4) in flow cytometry applications. It consists of the IL-4 antibody labeled with the tandem dye PE-Cyanine7, which enables simultaneous detection of IL-4 expression in cells.

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IL-4-PE (23 citations) PE-Cyanine7 (23 citations)

4 protocols using il 4 pe cyanine7

ELISPOT and Flow Cytometry Analysis of IFN-γ, IL-4, and TNF-α Production

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Spleen lymphocytes were seeded into anti-mouse IFN-γ mAb pre-coated 96-well plates at 2 × 10⁵ cells (100 μL/well) and stimulated with 10 μg/mL purified protein for overnight incubation; phorbol 12-myristate 13-acetate (PMA) was used as a positive control. The ELISPOT assay was performed using a mouse IFN-γ ELISPOT Kit (Dakewe) according to the manufacturer's instructions (Ranieri et al., 2014 ). Spots were counted using an immunospot reader (Cellular Technology Limited, USA).
Spleen lymphocytes (2 × 10⁶ cells) were firstly stimulated for 2 h with 10 μg/mL of purified E1-DIII + NS1-2 and E4-DIII + NS1-3 protein as a specific antigen. Then the treated cells were continue cultured in the presence of 10 mg/mL monensin and brefeldin A in complete RPMI 1640 (Gibco) overnight. Stimulated cells were first incubated and stained with Fixable Dye eFluor 506 and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience), CD45-APC-Cy7, and CD4-FITC (BD Biosciences) surface markers. Then, cells were fixed in permeabilization buffer (Thermo Fisher Scientific) and stained with IFN-γ-PE-eFlour 610, IL-4-PE-Cyanine7, and TNF-α-APC (eBiosciences). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.

He L., Sun W., Yang L., Liu W, & Li J. (2022). A multiple-target mRNA-LNP vaccine induces protective immunity against experimental multi-serotype DENV in mice. Virologica Sinica, 37(5), 746-757.

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Intracellular Cytokine Staining and Flow Cytometry

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For staining of intracellular IFN-γ, FOXP3, IL-4 and IL-17A, cells were stimulated for 5–6 h with Cell Stimulation Cocktail (plus protein transport inhibitors, 500 ×, eBioscience), which is a cocktail of phorbol 12-myristate 13-acetate (PMA) and ionomycin. The cells were harvested, washed twice with PBS, and analyzed for the presence of Treg and Th17 cells. The cells were then fixed, permeabilized, and stained with IFN-γ-PerCP, FOXP3-APC, IL-4-PE-Cyanine7 (11B11, eBioscience) and IL-17A-PE antibodies. Fluorescence signals were detected using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed by using FlowJo (Tree Star, Ashland, OR, USA) software.

Ren S., Zhang X., Guan H., Wu L., Yu M., Hou D., Yan Y, & Fang X. (2021). Lactobacillus acidipiscis Induced Regulatory Gamma Delta T Cells and Attenuated Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 12, 623451.

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Quantifying Murine T Cell Phenotypes

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To analyze PD-1 expression in CD4⁺ T cells, the cells were stained with anti-mouse CD3-antigen-presenting cell (APC) (clone 17A2, eBioscience, Waltham, MA, USA), CD4-FITC (clone GK1.5, eBioscience), PD-1-PE (clone J43, eBioscience). To detect intracellular cytokine expression, T cells from each mouse were stimulated with 2 µl/ml. Cell Activation Cocktail (with Brefeldin) (BioLegend, San Diego, CA, USA) in complete RPMI 1640 medium for 6 h at 37°C in 5% CO₂, then collected and surface stained with CD3 and CD4. The cells were washed, fixed, and permeabilized with cytofix/cytoperm buffer (BD Pharmingen), then intracellularly stained with anti-IFN-γ PE-Cyanine7 (clone XMG1.2), IL-4 PE-Cyanine7 (clone 11B11), and IL-17A PE-Cyanine7 (clone eBio17B7), or rat IgG1 and IgG2a isotype antibody (clone eBRG1, all from eBioscience) as control, respectively. To determine Tregs, the CD4⁺ cells were surface stained with anti-mouse CD3-PerCP (clone 17A2), CD4-FITC (clone RM4-5), and CD25-APC (clone PC61.5) in a Mouse Regulatory T cell Staining Kit (eBioscience). The cells were then permeabilized with cold Fix/Perm Buffer, and stained with anti-mouse Foxp3-PE (clone FJK-16s) or rat IgG2a isotype control antibody (clone eBR2a). Following immunofluorescence staining, samples were analyzed on a Flow cytometer (BD) using flowjo software (TreeStar). The cells were gated on CD3⁺ CD4⁺ T cells.

Cheng Y., Zhu X., Wang X., Zhuang Q., Huyan X., Sun X., Huang J., Zhan B, & Zhu X. (2018). Trichinella spiralis Infection Mitigates Collagen-Induced Arthritis via Programmed Death 1-Mediated Immunomodulation. Frontiers in Immunology, 9, 1566.

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Characterization of Antigen-Specific T Cell Responses

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Antigen-specific CD4⁺ and CD8⁺ T lymphocyte immune responses were characterized by the ICS assay. In brief, mouse spleens were removed under aseptic conditions and stimulated with the RBD of the S protein (10 μg/mL, Sino Biological, China). Then, the cells were incubated with GolgiPlug (BD Biosciences, USA), incubated and stained with Fixable Dye eFluor 506, and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience, USA), CD45-APC-Cy7, and CD4-FITC (BD Biosciences, USA) surface markers. The cells were then fixed in permeabilization buffer (Thermo Fisher Scientific, USA) and stained with IFNγ-PE-eFlour 610, IL4-PE-Cyanine7, and TNFα-APC (eBiosciences, USA). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.

Sun W., He L., Zhang H., Tian X., Bai Z., Sun L., Yang L., Jia X., Bi Y., Luo T., Cheng G., Fan W., Liu W, & Li J. (2021). The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice. Signal Transduction and Targeted Therapy, 6, 340.

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